Identification of important genes related to HVSMC proliferation and migration in graft restenosis based on WGCNA

The great saphenous vein is the most commonly used vessel for coronary artery bypass grafting (CABG), but its use has been associated with a high restenosis rate at 10-year follow-up. This study sought to determine the key genes associated with vein graft restenosis that could serve as novel therapeutic targets. A total of 3075 upregulated and 1404 downregulated genes were identified after transcriptome sequencing of three pairs of restenosed vein grafts and intraoperative spare great saphenous veins. Weighted gene co-expression network analysis showed that the floralwhite module had the highest correlation with vein graft restenosis. The intersection of the floralwhite module gene set and the upregulated gene set contained 615 upregulated genes strongly correlated with vein graft restenosis. Protein–protein interaction network analysis identified six hub genes (ITGAM, PTPRC, TLR4, TYROBP, ITGB2 and CD4), which were obtained using the STRING database and CytoHubba. Gene Ontology term and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses showed that the common hub genes were mainly involved in the composition of the cell membrane; in biological processes such as neutrophil degranulation, receptor binding and intercellular adhesion, innate immune deficiency; and other signaling pathways. Finally, ITGB2 was selected as the target gene, and its expression was verified in tissues. The results showed that ITGB2 was significantly overexpressed in occluded vein grafts. To study the function of ITGB2 in HVSMCs, primary HVSMCs were cultured and successfully identified. EdU incorporation, wound healing and transwell assays showed that ITGB2 silencing significantly inhibited the proliferation and migration of HVSMCs stimulated by PDGF-BB. Overall, our study provides a basis for future studies on preventing restenosis following CABG.

Coronary artery bypass grafting (CABG) remains the gold standard for treating patients with complex multivessel coronary artery disease and/or left main disease, diabetes or reduced left ventricular function, according to US and European guidelines 1,2 .Saphenous vein grafts (SVGs) are the most frequently used conduits for CABG, but their use is associated with a 10-year vein graft restenosis rate of 40-50% 3 .However, the mechanism of vein graft restenosis remains unclear, although it is widely thought to be related to multiple factors and mechanisms that cause intimal hyperplasia (IH), mainly involving the proliferation and migration of human vein smooth muscle cells (HVSMCs).Therefore, it is essential to further study the molecular mechanisms of IH following CABG.
Bioinformatics is a discipline based on integration of life sciences and computer sciences that involves the collection, processing, storage, transmission, analysis and interpretation of biological information 4 .Regarding currently available Gene Expression Omnibus (GEO) datasets associated with graft restenosis, most studies with uploaded data were on plasma from patients with in-stent restenosis, and three studies were about grafts in animals [5][6][7] .At this time, no studies about vein graft restenosis in human samples are included.
Herein, clinical samples of occluded vein grafts were collected for transcriptome sequencing and bioinformatics analysis to screen for key genes or proteins affecting the occurrence of vein graft restenosis.We found that

WGCNA
In our study, WGCNA was conducted with the R package "WGCNA".Clustering of the patients is shown in Supplemental Fig. 1, where red represents patients with occluded grafts.The key step in WGCNA is the selection of the soft-thresholding power.In our study, the soft-thresholding power was identified by network topology analysis.For WGCNA of vascular restenosis datasets, the soft-thresholding power was 12, and the lowest power of the scale-free topology fitting index was 0.8 (Fig. 3A).A hierarchical clustering tree of all genes in the vascular restenosis database was produced, and 10 important modules were generated (Fig. 3B).Moreover, the dendrogram and heatmap of the genes showed no significant difference in interactions among the modules, demonstrating a high degree of independence between these modules (Supplemental Fig. 2).The floralwhite module had the highest correlation with the of occluded graft status, with a correlation coefficient of 0.94 and a P value of 0.006 (Fig. 4).

Upregulated DEGs and protein-protein interaction network analysis
The Venn diagram of the DEGs in the floralwhite module and the upregulated DEGs is shown in Supplemental Fig. 3 and contains 615 genes.
The PPI network of the upregulated DEGs in the floralwhite module was constructed by the STRING online database and visualized by Cytoscape software (Fig. 5).Eleven algorithms were independently used to identify hub genes, and the 6 hub genes identified by the most algorithms were obtained: ITGAM, PTPRC, TLR4, TYROBP, ITGB2 and CD4.A summary of the hub genes is shown in Table 2.  GO and KEGG analyses of the common hub genes were performed to improve our understanding of the biological functions of these genes.GO analysis of the common hub genes by DAVID showed significant enrichment in components associated with the cell membrane, and the enriched biological processes included neutrophil degranulation and receptor binding, etc. (Fig. 6A).KEGG enrichment analysis showed significant enrichment in the intercellular adhesion pathway, innate immune deficiency pathway and other signaling pathways (Fig. 6B).

Validation of ITGB2 expression in occluded grafts
Based on the literature and the GO and KEGG analysis results, we finally selected ITGB2 as the target gene, and the expression of ITGB2 in occluded vein grafts and intraoperative spare great saphenous veins was validated by RT-qPCR and WB analyses.The results showed that ITGB2 expression in occluded vein grafts was significantly higher than that in intraoperative spare great saphenous veins (Fig. 7A-C).

Silencing of ITGB2 significantly decreases HVSMC proliferation and migration stimulated by PDGF-BB
To study the function of ITGB2 in vessels, primary HVSMCs were generated as previously described and characterized by an immunofluorescence assay (Supplemental Fig. 4).To investigate whether the proliferation and migration functions are regulated by ITGB2, we conducted EdU incorporation, wound healing and transwell assays.First, primary HVSMCs were transduced with si-ITGB2, and the knockdown efficiency was determined (Supplemental Fig. 5).Silencing of ITGB2 significantly decreased cell proliferation by approximately 50% in both the no-stimulation group and PDGF-BB group (Fig. 8A and Supplemental Fig. 6).Likewise, si-ITGB2 inhibited cell migration and invasion compared with that in the si-con group (Fig. 8B-D and Supplemental Fig. 7).Together, these results showed the function of ITGB2 in the proliferation and migration of HVSMCs.Silencing of ITGB2 may inhibit the PDGF-BB-stimulated proliferation and migration of HVSMCs.

Discussion
Abnormal proliferation of HVSMCs is a major cause of cardiovascular diseases and conditions, such as atherosclerosis, graft restenosis and aneurysm.To our knowledge, no study has assessed graft restenosis with human samples.In the present study, occluded vein grafts and intraoperative spare great saphenous veins were obtained from redo-CABG patients for transcriptome sequencing, and hub genes (ITGAM, PTPRC, TLR4, TYROBP, ITGB2 and CD4) were identified by bioinformatics analysis.GO and KEGG enrichment analyses revealed that the hub genes were associated with cell membrane components, intercellular adhesion and innate immune deficiency.
Atherosclerosis is observed during the late stage of graft restenosis and in the native coronary artery.Atherosclerosis is widely thought to result from abnormal lipid metabolism and chronic inflammation 8 .The ITGAM (also called CR3A, MO1A, CD11B, MAC-1, MAC1A, and SLEB6) gene, located on chromosome 16p11.2,encodes integrin-α M, which is an essential adhesion molecule and might promote the development and progression of abdominal aortic aneurysm by mediating the endothelial cell adhesion and transendothelial migration of circulating monocytes/macrophages 9 .One study showed that CD40L can interact with integrins to elicit monocyte adhesion and migration, representing a novel mechanism of inflammatory signaling in atherosclerosis 10 .TYROBP (also called DAP12, KARAP, PLOSL, and PLOSL1) encodes a transmembrane signaling polypeptide that is a type of transmembrane receptor ubiquitously expressed in macrophages/monocytes, natural killer (NK) cells and neutrophils 8 , and a recent study found that TYROBP promotes atherosclerosis 11 .In recent years, the involvement of NK cells has been documented in various inflammatory responses and early atherosclerosis 12 .Wang et al. revealed that atherosclerotic plaques in APOE mice exhibited high expression of TYROBP 13 .Vein graft restenosis is a chronic process involving atherosclerosis and inflammation.Consistent with these observations, we identified two genes significantly related to these processes.
PTPRC (also known as CD45) is an essential surface protein on hematopoietic and immune cells 14 .PTPRC controls immune function by regulating lymphocyte survival, cytokine responses, and TCR signaling 14 .Deficiency or altered expression of PTPRC is associated with various diseases, including leukemia and lymphoma 15 .PTPRC exerts effects through many mechanisms, including modulating apoptosis and cell survival 14 .In the present study, in silico analysis showed that PTPRC is related to vein graft restenosis, possibly through control of immune function and apoptosis.
Based on the literature, ITGB2 (also known as CD18) was selected as the target gene.The results showed that the expression of ITGB2 in occluded vein grafts was significantly higher than that in intraoperative spare great saphenous veins.An increasing body of evidence suggests that ITGB2 mutation may cause leukocyte adhesion defect type 1 (LAD-1) 16,17 .In recent years, it has been found that ITGB2 mediates a metabolic switch in cancerassociated fibroblasts, promoting oral squamous cell carcinoma proliferation 18 .In a study on myocardial infarction, overexpression of ITGB2 increased the migration and improved the engraftment of adipose-derived stem cells and augmented angiogenesis 19 .Sequencing of carotid atherosclerosis samples showed that ITGB2 was a key gene 20 .The above studies suggest that ITGB2 is tightly associated with the proliferation and migration of cells, in accordance with our results.Next, we found that silencing ITGB2 inhibited the proliferation and migration of HVSMCs.
In summary, our study demonstrates that the key genes related to graft restenosis include ITGAM, PTPRC, TLR4, TYROBP, ITGB2 and CD4.Moreover, ITGB2 knockdown can reduce the proliferation and migration of HVSMCs, a finding that offers novel insights into the prevention of restenosis following CABG.

Occluded vein grafts and great saphenous veins
In the present study, a total of fifteen pairs of occluded vein grafts and intraoperative spare great saphenous veins were obtained from patients undergoing clinical redo-CABG; three pairs were used for transcriptome sequencing, and the remaining pairs were used for experimental validation of the hub genes.In addition, the intraoperative spare great saphenous veins of CABG patients were used for primary HVSMC culture.The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013).The study was approved by the ethics committee of Tianjin Chest Hospital (No. IRB-SOP-016(F)-001-02), and informed consent was obtained from all individual participants.

Identification of DEGs from our sequencing data files
After transcriptome sequencing, we obtained the transcriptome expression profiles of three pairs of vessels.The expression profiles were normalized with the R package "limma".The differentially expressed genes (DEGs) were screened by the R package "limma" based on the cutoff criteria of |log 2 FC|> 1 and P value < 0.05.

Weighted gene co-expression network analysis (WGCNA)
WGCNA was performed with the R package "WGCNA" (https:// cran.r-project.org/ web/ packa ges/ WGCNA/ index.html) 21,22 .First, a co-expression network containing all genes was constructed, and the 20% of genes with the highest variance were used for further analysis.Samples were used to construct the adjacency matrix.Then, the adjacency matrix was transformed into a topological overlap matrix (TOM).Genes were classified into different modules by measurement of differences based on the TOM.In this study, we set the minimal gene module size as 30 and the threshold to merge similar modules as 0.25 to explore modules significantly correlated with clinical traits.

Identification of upregulated DEGs in the floralwhite module and construction of protein-protein interaction (PPI) networks
First, a Venn diagram was used to determine the intersection of the set of genes in the module with the highest correlation with clinical traits and the set of upregulated DEGs to identify upregulated DEGs in the floralwhite module; this was performed with FunRich, a biological analysis software (http:// www.funri ch.org/).The STRING database (http:// string-db.org/ 23 is widely recognized to collect, store and integrate publicly available sources of protein-protein interaction information and supplement these sources through computational predictions.In this study, PPI networks of the upregulated DEGs in the floralwhite module were analyzed through the STRING database (confidence score > 0.9) 23 .Subsequently, the PPI networks were visualized and analyzed with Cytoscape software and the cytoHubba plugin.All algorithms were applied to screen for hub genes.The top 6 genes were regarded as hub genes.

Functional annotation of the hub genes
The Gene Ontology (GO) database is a comprehensive resource of calculable knowledge about the functions of genes that is widely used by the biomedical research community to analyze omics and related data 24 .The Kyoto Encyclopedia of Genes and Genomes (KEGG) database has been reported to link genomic information with higher-order functional information 25 .In addition, the Database for Annotation, Visualization and Integrated Discovery (DAVID, https:// david.ncifc rf.gov/) provides a comprehensive set of functional annotation tools for investigators to understand the biological meaning behind large lists of genes 26 .In our study, GO and KEGG analyses of the hub genes were performed with the online DAVID tool (genes with the 10 highest −log 10 p values).

Real-time quantitative PCR (RT-qPCR)
Total RNA was isolated from the occluded vein grafts and intraoperative spare great saphenous veins of patients using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol.Total RNA from primary HVSMCs was isolated using a Takara reagent according to the manufacturer's protocol (Takara, Shiga, Japan).Complementary DNA (cDNA) was reverse transcribed from 2 μg of RNA by using the Takara reverse transcription system (Takara), and real-time PCR was performed with SYBR Green mix on the 7500 real-time PCR system (Applied Biosystems; ABI, Waltham, Ma, USA).ITGB2 expression was normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression, and relative expression levels were calculated with the 2-ΔΔCT method.

Figure 1 .
Figure 1.The methodology of the present study.

Figure 3 .Figure 4 .
Figure 3. Determination of the soft threshold of the gene coexpression network and module generation based on gene clustering dendrograms.(A) Determination of the soft threshold.(B) Module generation based on gene clustering dendrograms.

Figure 5 .
Figure 5.The PPI network of the DEGs_up gene set in the floralwhite module constructed via the STRING online database.

Figure 6 .
Figure 6.Functional and pathway enrichment analyses of the common hub genes.(A) GO analysis of the common hub genes.(B) KEGG analysis of the common hub genes.

Figure 7 .
Figure 7. Validation of ITGB2 expression in occluded grafts.(A) Relative expression of ITGB2 in occluded vein grafts, as measured by RT-qPCR.(B, C) Protein expression of ITGB2 in occluded vein grafts, as measured by WB.Control and C group represent the intraoperative spare great saphenous vein samples, and resentosis and T group represent the occluded vein grafts samples.Significance was indicated as *p < 0.05, ***p < 0.001.

Table 1 .
Characteristics of the fifteen redo-CABG patients.